PROJECT SUMMARY In eukaryotic cells, gene expression is controlled at multiple different layers. One of them is post- transcriptional gene silencing where microRNAs (miRNAs) bind target mRNAs in a sequence complementary manner and cause translational repression and/or deadenylation. In humans, miRNAs are loaded onto one of four Argonaute proteins (AGOs), forming a ribonucleoprotein complex terms `RNA-induced silencing complex (RISC)'. The target specificity of the RISC has been defined solely by the base complementarity between the miRNA (guide) and target strands. The loaded guide strand occupies part of the nucleic acid-binding channel between the AGO N-terminal and C-terminal lobes, while the remaining space serves as the composite target-binding channel. In this study, we hypothesize that the target specificity of the RISC is defined by the structure of the composite channel rather than just base complementarity, and thus that four human AGOs possess different target specificities due to their unique local structures. To validate this hypothesis, we will pursue the following specific aims. In Aim 1, cleavage assay and chemical probing will be used to determine how differently target strands are recognized in the presence and absence of the N- terminal lobe. In Aim 2, X-ray crystallography will be used to solve the structure of human AGO3-RISC. This structure, along with the previously determined ones, will enable us to identify local structures making their target-binding channels different from each other. RNA bind-n-seq experiments using wild type and its mutant lacking the identified unique local structure(s) will determine the target specificities conferred by the characteristic target-binding channel. In Aim 3, filter-binding assays and chemical probing will be used to elucidate the molecular mechanism by which a miRNA, miR-3191-5p, activates only AGO4 for binding to the internal ribosome entry site (IRES) of CACNA1A mRNA, and blocks its IRES-driven translation to prevent the neurological disease. Outcomes from this study will provide a new concept on the target specificity of the RISC, which is significant because beyond canonical gene silencing, many different cellular bioprocesses are regulated by miRNAs.